The site of mouse mammary tumor virus RNA transcription initiation has been determined in the presence and absence of glucocorticoid hormone (dexamethasone), and found to be invariant. Molecular clones of the Mtv-1 locus (responsible for mammary tumor induction in C3Hf mice) have been characterized. Transfection of XC/TK- cells with litigated Mtv-1 DNA and TK DNA gives rise to TK+ transfectants that express the Mtv-1 cloned DNA, both as glucocorticoid-regulated RNA and hormonally-induced gp52 env antigen. Sequencing of the Mtv-1 5' long terminal redundancy (LTR) identifies one region (about 100 nucleotides) of major rearrangement compared with the exogenous C3H-S LTR; this region is presumably not involved in the hormone target area.